University of Groningen
Stapled peptides inhibitors Ali, Amina
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Chapter 1
Stapled Peptides Inhibitors: A New Window For Target Drug Discovery
Ameena M. Ali, Jack Atmaj, Niels Van Oosterwijk, Daniel G. Rivera, Matthew R. Groves, and Alexander Dömling
(Submitted to Computational and Structural Biology Journal, Accepted)
Abstract
Protein-protein interaction (PPI) is a hot topic in clinical research as protein networking has a major impact in human disease. Such PPIs are potential drugs targets, leading to the need to inhibit/block specific PPIs. While small molecule inhibitors have had some success and reached clinical trials, they have generally failed to address the flat and large nature of PPI surfaces. As a result, larger biologics were developed for PPI surfaces and they have successfully targeted PPIs located outside the cell. However, biologics have low bioavailability and cannot reach intracellular targets. A novel class -hydrocarbon-stapled α-helical peptides that are synthetic mini-proteins locked into their bioactive structure through site-specific introduction of a chemical linker- has shown promise.
Stapled peptides show an ability to inhibit intracellular PPIs that previously have been intractable with traditional small molecule or biologics, suggesting that they offer a novel therapeutic modality.
In this review, we highlight what stapling adds to natural-mimicking peptides, describe the
revolution of synthetic chemistry techniques and how current drug discovery approaches have been
adapted to stabilize active peptide conformations, including ring-closing metathesis (RCM),
lactamisation, cycloadditions and reversible reactions. We provide an overview on the available
stapled peptide high-resolution structures in the protein data bank, with four selected structures
discussed in details due to remarkable interactions of their staple with the target surface. We believe
that stapled peptides are promising drug candidates and open the doors for peptide therapeutics to
reach currently “undruggable” space.
1 Introduction
Drug discovery approaches targeting protein-protein interactions (PPIs) has been fast-tracked over the present decade to deliver successful new drug leads and opens an expansive range of new therapeutic targets that were previously considered “undruggable”. This acceleration in PPI-based drugs is due to improved screening and design technologies, shortening the time between drug discovery to drug registration and changing pharmaceutical economic delivery [1]. Moreover, most human diseases are underpinned by a complex network of PPIs, (for example hubs such as p53), which underscores the need to understand PPIs not only on a clinical level, but also on molecular level. In this respect, the “omics” such as, genomics, RNA, proteomics and metabolomics can accumulate huge volumes of data aiming at targeted and personalized medicine [2,3].
All of the data, in addition to structural and screening-based approaches, have significantly expanded our understanding on PPI interfaces that were previously highly challenging and difficult to target, as these interacting surfaces are shallow or flat, non-hydrophobic and large (1500-3000Å).
In addition, PPI surfaces differ in their shape and amino acid residue composition, particularly the hot spots that are essential during binding protein partners; making small-molecules entities unlikely as protein therapeutics [4–8]. Moreover, the discovery of innovative and drug lead molecules with the expected biological activity and pharmacokinetics is the main aim of medicinal chemistry. Therefore, the application of ‘follow-on’-based strategy has always been one of the most effective approaches that lead to promising bioactive molecules. Conformational restrictions or
“rigidification” is one of these strategies that has been widely used to overcome ligand flexibility, which suffer from entropic penalty upon binding to the target surface [9]. The restriction strategy has two major advantages: firstly, it could increase the potency of the drug-like agent by stabilizing a favorable binding conformation, reducing the entropic penalty on binding to the target and decrease its degradation by hindering metabolically labile sites or introducing a fused-ring structure; in addition to improve isoform selectivity or specificity toward targets. Secondly, controlling ligand confirmation could improve affinity on the atomic level without requiring additional interactions [9,10].
There are two types of drugs generally available on the market: traditional small-molecule drugs
with molecular weight of <500 Daltons and high oral bioavailability but low target selectivity; and
biologics that are typically >5000 Daltons (such as insulin, growth factors, erythropoietin (EPO)
and engineered antibodies) that have limited oral bioavailability, poor membrane permeability and
metabolic instability. As a result such medications are typically delivered by injection. However,
biologics have extremely high specificity and affinity for their targets due to the large area of
interaction with their targets [1,11]. Despite the success of both drug classes in treating different
diseases, there remains an opportunity to offer a class of molecules to fill the gap in molecular
weight between the existing two classes (Small molecules <500...Peptides…Biologics >5000
Daltons) and merge some of the advantages of small-molecules and biologics in terms of oral
bioavailability, cell penetration and cheaper manufacturing costs. This class could be considered to
be a next generation therapeutic class that precisely targets PPIs and is based upon hydrocarbon-
stapled α-helical peptides. Figure 1 represents the three classes of targeted drugs based on their molecular weight.
In this review we will focus on hydrocarbon-stapled α-helical peptides and their use as potential drugs. Hydrocarbon α-helical peptides are synthetic mini-proteins locked into their bioactive α- helix secondary structure by site-specific insertion of a synthetic chemical staple linker or “brace”.
Stapled peptides show a greatly improved pharmacologic performance, increased affinity to their target, resistance to proteolytic digestion, and afford high levels of cell penetration via endocytic vesicle trafficking [5,12–14].
In this review we will discuss what stapling adds to this class of inhibitors in terms of stability, bioactivity and cell penetration, the chemistry behind peptide stapling and provide an overview on some selected successful examples of peptide-based drugs to underline their importance. Lastly, we will underline four exclusive stapled-peptides targeting PPIs, in which their staple makes an intimate interaction with the target interface, in order to reveal the role of stapling on peptide binding and their inhibition of PPIs.
2 Why Stapled Peptides?
Helical peptides are one of the two main secondary structural elements in PPI interfaces, (in addition to β-sheets) and play a central role in protein function within the cell. Often these elements are not stable in conformation in the absence of a complete protein fold. Additionally, peptides are sensitive to proteolysis by peptidases reducing their half-life (down to minutes), impacting their ability to penetrate cell membranes – all of which makes native peptides poor drug candidates [14–
16]. Notwithstanding, one main feature that makes peptides good drug candidates is their ability to bind large and relatively flat target surfaces efficiently and specifically, which is a requirement in
NH
SO O HO
O NH2 CN
O NH
Ph CO2Me
NH2Me
Molecular Weight (Da)
Size
<500 Da >5000 Da
Small-molecules
Hydrocarbon
α-helix peptides Biologics
Figure 1: The three classes of targeted medicines. The traditional small- molecules inhibitors were the first class discovered to inhibit different PPIs surfaces with MW of <500 Da and high bioavailability. Most of the biologics, the second class of PPI targeted molecules, have a MW of more than 5000 Da (eg. antibodies and growth hormones) aimed to overcome a broad range of diseases. Stapled α-helix peptides as a class address this gap in MW between small molecules and biologics, aiming to combine the oral bioavailability of small-molecules with the high specificity of biologics toward the target protein.
the majority of intracellular therapeutically relevant PPIs. This makes peptides as an attractive target for drug development and enables their transition into the clinic [5,17]. The use of therapeutic peptides has grown explosively over the last three decades, covering areas such as metabolic diseases, oncology, and cardiovascular diseases [18]. From a dataset that was collected recently on March 2018 and based on previously released database report by Peptide Therapeutics Foundation, of 484 therapeutic peptides, 60 have been approved in the United States, Europe, and/or Japan, 155 peptides are in clinical development and 50% are currently in Phase II studies (Figure 2) [18].
Massive efforts and optimizations have been conducted in order to overcome the limitations above.
To impose a peptide α-helix conformation (thereby improving their binding affinity toward their target protein) non-native amino acids were used in the peptide that lie on the same helix face.
These non native amino acids are then linked together or “stapled” through side-chains that can be covalently bonded [16,19].
In order to address a second issue, to synthesize peptides with resistance toward proteases, non- peptide (such as cyclic tripeptides, heterocyclic or other organic constraints) are inserted into a peptide sequence to maintain the peptide backbone in a linear saw-toothed strand structure [20–23].
These chemical modifications have evolved over time since the first all-hydrocarbon stapling by Verdine and colleagues in 2000, who produced a large series of α, α-disubstituted non-natural amino acids bearing olefin tethers (Figure 3a). His work was an extension of Blackwell and Grubbs, who were the first to use Grubbs catalysts to make a cross-link between O-allylserine residues on a peptide template (Figure 3b). Walensky provided the bridge between chemistry and biology by generating hydrocarbon- stapled BH3 peptide helices, targeting BCL-2 homology 3 domains responsible for the interactions of BCL-2 family proteins that mainly regulate cellular life and death
Figure 2: Statistical representation of therapeutic peptides until March 2017. The numbers are indicated in percentage at each category with a total number of 484 medicinal peptides that were produced with development activity regulatory approval from major pharmaceutical markets as, the United States, Europe, and Japan. From these peptides 12% were approved, while 32% are in clinical trials and further classified as phases I, II, III and pre-registered. The highest percentage (54%; “Discontinued”) category encompasses peptides terminated before approval. The lowest percentage 2% is the
“Withdraw” category that refers to previously approved peptides that are no longer available in the market [18].
resistance to proteolysis, but also high cellular permeability [19,24,25]. The details of stapled- peptide chemical synthesis will be discussed in detail in section 3.
Interestingly, peptides could be differentiated from proteins by their size (50 amino acids or less) but have similar specificity toward their targets as biologics. However, peptides are more potent binders to PPIs interfaces, because of their ability to bind large protein surfaces with great selectivity and less toxicity when compared to small molecule drugs, which often produce toxic metabolites. In contrast to small molecules, peptides are degraded into amino acids, which are in turn not toxic or harmful for cells [1,26]. Furthermore, peptides have lower manufacturing costs and are more stable at room temperature (unlike recombinant antibodies and engineered proteins).
Finally, as non-natural amino acids are the building blocks of peptides, the opportunity to produce diverse scaffolds with modified chemical and functional properties is available [27,28].
Structural knowledge of the target PPIs and mutagenesis data for residues at or near the binding interface are necessary to achieve a successful interruption of PPI partner proteins in vivo. Peptide design is based upon the ligand-target pair, in that the ligand retains its α-helical motif and is docked into shallow cleft surface of the target protein. Thus, stapled peptide inhibitors represent
“dominant-negative” versions of the docking helix [5]. The peptide is then optimized by sequence modification “or stapling” to improve cell penetration and peptide efficacy to compete with the intracellular ligand protein, and it is crucial to position the cross-linking amino acids in such way that the targeted interface remains intact [5]. After evaluating the cellular uptake of the stapled
Figure 3: Ruthenium-catalyzed ring-closing metathesis (RCM) reaction for peptides stapling was a) published for the first time by Verdine and Schafmeister in 2000 by engaging α,α- disubstituted non-natural amino acids harboring all-hydrocarbon tethers [19]. Their work was a continuation of b) Blackwell and Grubbs work in 1998 [24]; who performed ruthenium- catalyzed olefin metathesis for macrocyclisation of synthetic peptides using a pair of O-allylserine residues in a metathesis reaction.
O O
O O
CH3
H3C CH3 H3C
O-allyl Serine Residues (RCM) α,α-disubstitution, Olefin tether (RCM)
a) b)
peptides using live confocal microscopy, a broad spectrum of cellular and in vivo studies are applied to examine the therapeutic activity of the stapled peptides toward their targets. A flow-chart in Figure 4 summarizes the development process of therapeutic peptides for biological study, from virtual design to in vivo mouse model analysis. Examples of stapled peptide created through the use of high-resolution structures are SAHBA, based on BH3 domain of proapoptotic BID protein [25], SAH-p53, based on the p53-MDM2 interaction interface [29], SAH-gp41 double stapling peptide, targeting the HIV-1 virus and Enfuvirtide, the first decoy HR2 helix fusion inhibitor [30]. If the proteins involved in the PPIs of interest have no previous structures, Ala-scanning or residue conservation “in situ mutagenesis” can be used as a starting point to position the staple. If this information is also not available, then synthesizing and screening all stapling positions is advisable [5].
3 Chemical Synthesis of Stapled Peptides
As the synthesis of bioactive-stapled peptides started to widen, the approaches used also branched and allowed stapled peptides to be applied for various purposes such as target binding analyses, structure determination, proteomic discovery, signal transduction research, cellular analyses, imaging, and in vivo bioactivity studies [31]. Solid-phase peptide synthesis (SPPS) is a standard and commonly used chemical procedure to synthesize α-helix peptides. The first required entity to start stapled peptides synthesis is a stock of non-natural amino acids building blocks with a variable length of the terminal olefin tethers. The choice of the non-natural amino acids will define the length, structure and the chemical functionalities of the stapled linker [14,32]. The helix backbone
Figure 4: Workflow of all hydrocarbon-stapled peptides generated for biological investigation. Computational designation of the peptides including in-situ mutagenesis to screen all possibilities based on previous reported structures, followed by in vitro biochemical, structural, and functional studies compromising peptides binding affinities measurements toward the target protein interface utilizing biophysical assays and crystallization trials. Potent binder peptides will be further tested for their cellular uptake and permeability using live confocal microscopy. Lastly, successful peptides are subjected to a broad spectrum of cellular and in vivo analyses, using mouse models of the studied disease.
amino acids are protected with a base-labile fluorenylmethoxycarbonyl (Fmoc) to obtain N- α - Fmoc-protected amino acids, which are often offered with acid-labile side chain protecting groups that vary between the 20 amino acids. The side chain protecting groups of each amino acid for standard SPPS of stapled peptides are indicated in Table 1. After the synthesis of non-natural amino acids and peptide elongation during SPPS; ring-closing metathesis (RCM) of the stapled is performed.
Table1: The acid-labile side chain protecting groups used in SPPS synthesis of stapled peptides
Amino acid Three letters-code Side chain protecting group
Alanine Ala N/A
Cysteine Cys Trityl (Trt)
Aspartic acid Asp tert-Butyl (OtBu)
Glutamic acid Glu tert-Butyl (OtBu)
Phenylalanine Phe N/A
Glycine Gly N/A
Histidine His Trityl (Trt)
Isoleucine Ile N/A
Lysine Lys tert-Butoxy (Boc)
Leucine Leu N/A
Methionine Met N/A
Asparagine Asn Trityl (Trt)
Proline Pro N/A
Glutamine Gln Trityl (Trt)
Arginine Arg Pentamethyldihydrobenzofurane (Pbf)
Serine Ser tert-Butyl (OtBu)
Threonine Thr tert-Butyl (OtBu)
Valine Val N/A
Tryptophan Trp tert-Butoxy (Boc)
Tyrosine Try tert-Butyl (OtBu)
SPPS has been automated using Fmoc chemistry to become an efficient and reliable method to yield hydrocarbon-stapled peptides of single or double stapling with different functionalities and experimental applications. However, SPPS has two main complications: First, efficiency is limited in longer peptides (>50 residues). These are more usually expressed using recombinant DNA technology, due to the unavailability of the N-terminal amine of the non-natural amino acids (mostly after naturally bulky residues like arginine or β- branched amino acids (valine, isoleucine, and threonine)). Additionally, extension of deprotection and coupling times with fresh reagent may be required in the synthesis of larger peptides. The second complication is that cross-reaction or progressive inaccessibility of the N-terminus due to on-resin aggregation could occur [31–33].
Initial screening of different types of stapling is required if structural-based knowledge is not
available. As indicated previously in section 2, prediction software can suggest the peptide α-helix
template, then a group of constructs with differentially localized staples can be generated to
determine the optimal staple placement. However, if the target PPIs interface is structurally well
characterized, this structural data can be used for computational docking and designing of the
desired template peptide to generate a panel of peptides with diverse stapling type and position.
Stapling techniques could be divided into one-component or two-component stapling techniques, based on the side-chain linking reaction. During one-component stapling a direct bond will be formed between two non-natural amino acids side-chains, whereas two-component stapling involves a separate bifunctional linker to connect the side-chains of two non-natural amino acids [14]. The most commonly used technique for stapling is the one-component stapling technique - employing S-pentenylalanine at i,i+4 positions for one turn stapling or combining either R- octenylalanine/S-pentenylalanine or S-octenylalanine/R-pentenylalanine at i,i+7 positions. Other spacings for stapling were also accomplished upon chemical optimization, including i,i +3 and i,i+11 [14,31,32,34]. The common stapling positions are shown in Figure 5.
There are several chemical procedures to enclose or stabilized the all-hydrocarbon linker into α- helix peptide such as, ring-closing metathesis, lactamisation, cycloadditions, reversible reactions and thioether formation. A brief summary for each methodology and some literature examples is provided below.
Figure 5: a) The common stapling insertion positions for α-helix peptides. Combinations of two non-natural amino acids S5, R5, S8 and R8 are used for different positions of stapling the hydrocarbon linker. Employing S5/S5 at position i,i+4 is the most common stapling position on the same face of helix turn. For i,i+7 position, two combinations could be applied either S8/R5 or S5/R8. Synthetic chemistry evolved to introduced i,i+3 and i,i+11 as new possible positions for stapling in addition to double-stapling. b) The structures of the four designed amino acids used to introduce all-hydrocarbon staples into peptides.
All possess an α-methyl group (Me) and an α-alkenyl group, but with opposite stereochemical configuration and different length at the alkenyl chain.
a)
i,i+4 i,i+3
i,i+7 i,i+11
H2N
OH O (R)
Me
R8 R5
H2N
OH O (S)
Me
S8 S5
H2N
OH O (R)
Me
H2N
OH O (S)
Me
b)
3.1 Ring-closing metathesis (RCM)
Blackwell and Grubb were the first to apply alkene ring-closing metathesis as a peptide stapling method. They described solution-phase metathesis, followed by hydrogenation of hydrophobic heptapeptides containing either O-allyl serine or homoserine residues with i,i+4 spacing (Figure 6) [24]. Their study emphasized the feasibility of metathesis on helical peptide side-chain. Later in 2000, Schafmeister and his colleagues managed to conduct metathesis stapling using α,α- disubstituted amino acids carrying olefinic side-chains of different lengths and stereo-chemistry on solid phase prior to peptide cleavage from resin, producing a large series of α, α-disubstituted non- natural amino acids S5, R5, S8 and R8 bearing olefin tethers that were used for different stapling positions of the hydrocarbon linker as shown in Figure 5. The end products were a collection of i,i+4/7 peptides and they found that i,i+7 stapled peptides have higher helicity and stability over native and non-stapled peptides [19]. BID BH3 peptides that bind to BCL-2 family proteins are a successful product of metathesis stapling by Walensky et al. and they showed that the optimized stapled peptide has more stability than the native one, provoke apoptosis in leukaemia cells, and inhibit the growth of human leukaemia xenografts in mice [25]. p53-MDM2/MDMX dual inhibitor stapled peptides were reported by Sawyer and co-workers, who provided promising in vitro data for binding affinity, cellular activity and suppression of human xenograft tumours in animal models [35]. These findings are the basis of p53 optimized stapled peptides that have enter clinical trials.
Further, Verdine et.al introduced a unique form of multiple stapling, called stitches, in which two hydrocarbon staples immediately follow one another. This technique requires the use of the amino acid bis-pentenylglycine (B5) that forms a junction between the two staples and emerges from a common residue in the peptide. There are many possible combinations of stereochemistry and linker length in such a system. Various stitch combinations were studied rigorously and two systems, i,i + 4 + 4 (S5 +B5 +R5) and i,i+4+7 (S5 +B5 +S8), appeared the most effective for helix stabilization. A peptide with the latter stitch construction was found to have superior helicity and cell penetration compared with an i,i + 7 stapled analogue [36].
Optimization and extensive development in hydrocarbon stapling approach allow stapling at i,i+3/4/7 spacings. Regardless of the many examples in literature of successful hydrocarbon stapling, there is no guarantee that stapling will enhance peptide stability, cell penetration and binding to the target. Extensive optimization is needed in order to discover a staple peptide with the desired features.
Figure 6: Figure 6: RCM or ring closing metathesis reaction for synthesis of the all-hydrocarbon stapled peptide reported by Schafmeister et al. 2000, which increase peptides helicity as found by circular dichroism (CD) [19].
Ac-EWAE NH AAAKFL HN AHA
( )6 ( )3
Ac-EWAE NH AAAKFL HN AHA
( )6 ( )3
3.2 Lactamisation
Stabilization of an α-helix can also be accomplished through side-chain intramolecular amide-bond formation between i,i+4 spaced amine- and carboxy-side chain amino acids. Lactamisation has been studied first by Felix and his group in 1988 [33], when they coupled Lys and Asp residues side- chains in growth hormone releasing factor short congener. Growth hormone helicity and activity were conserved post macrocyclisation, which were measured by NMR and circular dichroism (CD) (both methods that can be used for analyzing the secondary structure of peptides and proteins in aqueous solution) [34]. Since then, numerous studies applied lactamisation and amide linkage on different chain length and positions, with the intention to generate a stable helix for different systems. For example, a lactam stapling optimization study on penta/hexapeptides between Orn/Lys and Asp/Glu residues, carried out by Fairlie and co-workers (Figure 7) [35], examined the shortest possible peptide with α-helix reinforced structure in water. Subsequently, the Fairlie group applied their finding on different targets including inhibition of respiratory syncytial virus with double lactam-stapled peptide in 2010 with improved antibacterial activity. Another target was nociceptin hormone studied in the same year 2010, in which lactam-stapled peptide induced higher ERK phosphorylation in mouse cells and thermal analgesia [19]. Norton and co-workers also examined several Asp/Lys lactam-stapling combinations at i,i+4 position on µ-conotoxin KIIIA, a natural peptide from mice that acts as a potent analgesic by binding voltage-gated sodium channels (VGSCs), where they found that stapled peptides have different level of helicity and inhibitory activity on variable VGSC when examined in Xenopus laevis oocytes [36]. From a chemical prospective, lactam stapling is easier to obtain and incorporate due to proteogenic amino acids when compared to other stapling techniques, which required non-proteogenic amino acids. A drawback is that an extra orthogonal protecting group is needed for selective deprotection of the amine and acid functionalities prior to lactamisation. Another limitation of this technique should be mentioned, which is the lactamisation stapling of Lys and Asp residues. Stapling at these residues is only compatible with i,i+4 spacing with longer linkages that required modified amino acids with longer side-chains. From a biological point of view and based on a large number of studies on peptide lactamisation, this stapling technique can create therapeutic peptides with superior bioactivity and most of their targets are either extracellular or membrane-bound, suggesting that lactamisation stapling has no potential to improve cell penetration [12].
Figure 7: A Lactamisation study that was conducted by Fairlie and co-workers on penta and hexa-peptides in order to optimize lactam stapling between Orn/Lys and Asp/Glu residues. It wasn’t the first study for lactam optimization; however, the group was abled to systematically and quantitatively found the shortest peptide with retained helicity in water as judged by CD [39].
Ac NH C ARA NH C
O O
NH2 ( )4
O HO
Ac NH C ARA NH C
O NH
O O
3.3 Cycloadditions
Cu (I)-catalysed azide–alkyne cycloaddition (CuAAC) or the “Click” reaction is another mechanism of peptide stapling, it is also known as biocompatible ligation technique [41]. The first research group who applied CuAAC to generate α-helix structures between i,i+4 spacing within peptides were Chorev, D’Ursi and co-workers in 2010, based on parathyroid hormone-related peptide [42]. Subsequently, many groups used this type of stapling in order to determine the best linker length, including Wang and co-workers, who found that five methylene units were the optimum staple length to inhibit the oncogenic BCL9-beta-catenin PPI (Figure 8). A further significant result, reported by the same team, of the Click reaction was based on triazol-position screening along a peptide targeting the same oncogenic protein, beta-catenin, to generate a library of stapled peptides exhibiting different in vitro binding affinities and helicity [43]. Madden et al., used an unusual cycloaddition via UV-induction between tetrazoles and alkenes to hinder p53- MDM2/MDMX interaction. The stapling reaction took place between i,i+4 by exposing unprotected linear peptides to UV irradiation in solution, which resulted in stapled peptides displaying higher affinity toward MDM2/MDMX in a fluorescence polarisation assay (FP).
However, these peptides were not cell permeable. This problem could be overcome by modifying a number of the peptide amino acids to Arg, whereby cellular uptake and moderate p53 activity were achieved [44]. Generally, stapling with cycloaddition chemistry shows a promising future, in that triazol- stapled amino acids are accessible and CuAAC is well established. In the example of UV- induced reactions, the method is simple to apply but requires extra analysis that might affect applicability in other biological systems..
3.4 Reversible reactions
Using disulphide bridges between two Cys residues as stapling technique was first introduced by Schultz et al. at i,i+7 positions. The disulfide bridge was formed between D and L-amino acids having thiol-side chain, followed by the addition of acetamidomethyl (Acm) protecting groups, protection and oxidization with iodine (Figure 9). The helicity of disulfide-stapled peptides was higher when compared with the Acm-protected precursors, as displayed in CD spectroscopy [45].
Although disulfide stapling was the earliest reported stapling technique, little was concluded due to the instability of the disulphide stapled peptides in reducing environments, which restrict their application in intracellular targets. However, stapling with oxime linkages [46] and two-component bis-lactam and bis-aryl stapling techniques [47,48] were found to be superior to the analogous disulphide stapling. Recently, Wang and Chou demonstrated the possibility of stapling and macrocyclization using thiol-en between two Cys residues an α, ω-diene in high yields (an unsaturated hydrocarbon containing two double bonds between carbon atoms), which allowed stapling of both expressed/unprotected and synthetic peptides. This group applied their discovery to
Ac-LSQEQLEHR NH C NH C TLRDIQRMLF-NH2
O O
N3 ( )4
RSL
Figure 8: Optimized CuAAC-stapled peptide was successfully developed to inhibit the BCL9 oncogenic interaction. After screening different stapling length, Wang and co-workers concluded that five units of methylene was optimal stapled peptide for BLC9 inhibition [43].
Ac-LSQEQLEHR NH C NH C TLRDIQRMLF-NH2
O O
RSL NN
N
the p53-MDM2 PPI and successfully synthesized stapled peptides with both i,i+4 and i,i+7 linkages, applying this method in the stapling of large peptides and proteins. Development in reversible stapling is slow, but efforts in applying this method in biological dynamic covalent chemistry are under active investigation.
3.5 Thioether formation
The reaction between Cys thiol and alpha-bromo amide groups has been developed as a protocol for peptide stapling by Brunel and Dawson [49]. This linkage was designed to mimic the ring size of previously reported lactam staples, but a thioether link was hosted into gp41-peptide epitopes as an approach to establish an HIV vaccine. Successful staples were created in both i,i+3 and i,i+4 linkages and a peptide with i,i+3 stapling (Figure 10) demonstrated a higher helicity over unstapled and lactam-stapled peptides i,i+4. Moreover, after optimization the stapled peptide bound to a gp41-specific antibody (4E10) more effectively than the uncyclised peptide [50]. These findings illustrate the efficiency of thioether stapling with shorter distance i.e. i,i+3, while suggesting that lactam staples are more suitable for i,i+4 stapling.
Figure 9: Schultz and co-workers described an i,i + 7 stapling methodology using disulphide bridges between D and L amino acids bearing thiol-side chains. The amino acids were connected with acetamidomethyl (Acm) protecting groups, deprotected and then oxidised with iodine to give a disulphide stapled peptide. CD spectra of disulphide stapled peptides exhibited a high level of α-helicity in comparison to the Acm-protected precursors that were significantly less helical [45].
Ac-AAA NH C N
H C AAAKA-NH2
O O
( )4S S
AcNH NHAc
( )4
KAAAAK Ac-AAA NH C N
H C AAAKA-NH2
O O
S S
KAAAAK
Figure 10: Thioether stapling method was reported by Brunel and Dawson in 2005. They demonstrated the reaction of Cys thiol and alpha-bromo amide groups to report a i,i+3 thioether stapled peptide that inhibited HIV fusion using the gp41 epitopes as template for peptide synthesis [49].
H2N C N
H C ITNWLWKKKK-NH2
O O
SH HN
O
( )3 WF
Br
H2N C N
H C ITNWLWKKKK-NH2
O O
S HN
O
WF
( )3
4 Structural Insight of Stapled Peptides Target Protein-Protein Interaction (PPI) in the PDB
The number of peptides entering clinical trials has increased over the last 35 years, with an average peaking in 2011, when over 22 peptides/year were successful in entering clinical development [18].
This evolved from technology maturation and advances in synthetic chemistry and purification of peptides, in parallel with improvements in biophysical and molecular pharmacological methods.
However, there is a limited number of high-resolution structures of staple peptides in complex with their targets in the protein database (RCSB www.rcsb.org), as of July 2018 [51]. There are 67
“stapled peptides” structures, of which 58 are based on X-ray diffraction (83%) and 16 on NMR studies (17%). When limiting the analysis to Homo sapiens, our search found 43 structures, targeting a limited range of PPIs, of which the majority are of the p53-MDM2/MDMX interaction, the BCL-2 family (including the MCL-1 BH3 domain), estrogen receptor and human immunodeficiency virus type 1 (HIV-1). Other druggable interfaces of interest were kinases [52,53], insulin [54], tankyrase-2 [55], growth factor receptor-bound protein 7 (Grb7) [56], the Fc portion of human IgG [57], eIF4E protein [58] and transducin-like enhancer (TLE) proteins [59].
MDM2 and its homolog MDMX represent 18.6% of total X-ray structures in protein data bank, which indicates their importance as the main negative regulators of p53 (The Guardian of the genome) since it behaves as a hub protein [60]. This escalation in stapled peptide drug discovery has crossed over into the traditional focus upon endogenous human peptides to include a broader range of structures identified through medicinal chemistry efforts. Not surprising, today over 150 stapled peptides are in the active development of human clinical studies [18].
The analysis presented in Table (2) provides a list of the crystal structures belong to therapeutic stapled peptides, which mimic the native peptides in complex with their target protein interfaces.
The table also gives an overview of the PDB structure code, name of stapled peptide and
biophysical assays that are used to measure the binding dissociation constant (K
d) of the stapled
peptide to the target protein.
Table (2): List of structural-resolved stapled peptides in complex with PPI targets from RCSB-PDB
Target PDB ID Binding Assay Peptide Kd (nM) Ref.
X-ray NMR
Human MDM2/4 IYCR ITC p53-WT Residues (15-29) 600 [61] NA
3V3B FP SAH-p53-8 Stapled peptide 55 [62] NA
ITC 12
4UMN FP M06 Stapled peptide 63±17.8 [63] NA
5AFG FP E1 Stapled peptide 7.5±0.7 [64] NA
4UE1 FP YS-1 Stapled peptide 9.9±1.5 [65] NA
4UD7 FP YS-2 Stapled peptide 7.4±1.5 [65] NA
5XXK FP M011 Stapled peptide 6.3±2.9 [66] NA
5VK0 - PMI - [67] NA
5VK1 - PMI - [67] NA
MCL-1/ BCL-2 3MK8 FP MCL-1 SAHBD Stapled peptide 10±3 [68] NA
5C3F FP BID-MM Stapled peptide 153±12 [69] NA
SPR 107±29
5C3G SPR BIM-MM Stapled peptide 460±232 [69] NA
5W89 FP SAH-MS1-18 Stapled peptide 25±7 [70] NA
5W8F FP SAH-MS1-14 Stapled peptide 80±5 [70] NA
5WHI - BCL-1 Apo - [71] NA
5WHH Streptavidin pull-down D-NA-NOXA SAHB Stapled peptide - [71] NA
Estrogen Receptor 2YJD SPR SP1 ERβ/1.99 µM
αER/674
[72] NA
2YJA SPR SP2 ERβ/632
αER/352
[72] NA
5DXB SPR SRC2-SP1 530 [73] NA
5HYR SPR SRC2-SP2 42 [73] NA
5DX3 SPR SRC2-SP3 39 [73] NA
5DXE SPR SRC2-SP4 - [73] NA
5DXG SPR SRC2-SP5 - [73] NA
2LDA - SP2 - NA [72]
2LDC - SP1 - NA [72]
2LDD - SP6 ERβ/155
αER/75
NA [72]
5WGD - SRC2-LP1 - [74] NA
5WGQ - SRC2-BCP1 - [74] NA
Aurora-A 5LXM ITC Stapled TPX2 peptide 10 0.18 µM [73] NA
Tankyrase-2 5BXO FP Cp4n2m3 0.6±0.01 µM [55] NA
5BXU FP Cp4n4m5 2.8±0.1 µM [55] NA
Grb7 5D0J SPR G7-B4NS peptide 4.93±0.03 µM [56] NA
5EEL SPR G7-B4 peptide 0.83±0.006 µM [56] NA
5EEQ SPR G7-B1 peptide 1.5±0.01 µM [56] NA
Replication proteinA 4NB3 FP Peptide-33 0.022±0.005 µM [75] NA
eIF4E 4BEA SPR sTIP-04 Stapled peptide 5±0.7 [58] NA
FP 11.5±3.6
β-catenin 4DJS FP aStAx-35 13±2.0 [76] NA
hDcn-1 3TDZ ITC hCul1WHB : hDcn1P :
Acetyl-hUbc121-12(5:9 Staple)
0.15 µM [77] NA
Insulin 3KQ6 Receptor Binding Assays [HisA4, HisA8] insulin IGF-1R/
0.05±0.01 IR/125±18
[54] NA
ks-vFLIP 5LDE ITC spIKKƔ-Stapled peptide 30.4±3.8 µM [52] NA
TLE1 5MWJ ITC Peptide18 522±39.6 [59] NA
human IgG1 Fc 5U66 SPR LH1 ~1±0.5 mM [57] NA
Not all staples interact with the target protein surface via commonly known chemical interactions;
instead they can induce conformational changes to either the synthetic α-helix peptide or the target protein interface, specifically the amino acids residues involved in PPIs. These changes stabilize and fix the helical peptide in a potent binding mode within the target interface. A limited number of stapled peptides have different interactions with their intracellular targets, which contributes to their high specificity, stability and makes these peptides promising target therapies for human diseases.
Table (3) underlines the role of the stapled linker in binding to the target protein surface and indicates if it is involved in any interaction with the target surface residues via Van der Waals, hydrogen/disulfide bonds or 𝝅-𝝅 interactions. All of these interactions were inspected from the crystal structures of the stapled peptides in complex with the target protein surfaces. Examples of these peptides will be discussed extensively in the next sections to highlight the evolution of medicinal chemistry techniques.
Target PDB ID Binding Assay Peptide Kd (nM) Ref.
X-ray NMR
CaV β subunit 5V2P ITC AID-CAP Stapled peptide 5.1±1.6 [78] NA
5V2Q ITC AID-CEN Stapled peptide 5.2±1.5 [78] NA
NCOA1 5Y7W - YL-2 - [79] NA
Saccharomyces cerevisiae
5NXQ FP Sld5 CIP A2 0.32 ± 0.02 µM [80] NA
4HU6 - GCN4-p1(7b) - [81] NA
CRPs (Plants) 5NGN - Lyba2 - [82] NA
HIV-1 4NGH - SAH- MPER(671-683KKK)(q)pSer - [13] NA
4NHC - SAH-MPER(671-683KKK)(q) - [13] NA
4U6G - SAH-MPER(662-683KKK)(B,q) [13] NA
8HVP - Ua-I-OH 85548e - [83] NA
7HVP - JG-365 - [84] NA
2L6E Total buried surface NYAD-13 1 µM NA [85]
2JUK - GNB - NA [86]
1ZJ2 - HIV-1 frameshift site RNA - NA [87]
1PJY - HIV-1 frameshift inducing stem–loop
RNA
- NA [88]
Brevibacillus Bacteria 4OZK - LS - [89] NA
Zebrafish MDM2/X 4N5T Biacore ATSP-7041 MDM2/ 0.91
MDMX/ 2.31
[35] NA
Plasmodium falciparum 4MZJ - pGly[801-805] - [90] NA
4MZK - pGly[807-811] - [90] NA
4MZL - HSB myoA - [90] NA
XRMV 4JGS - ɣ-XMRV TM retroviral fusion protein - [91] NA
MPMV 4JF3 - β-MPMV TM retroviral fusion protein - [91] NA
Salmonella 1Q5Z - SipA - [92] NA
Synthetic collagen 3P46 - SS1 - [93] NA
EphA2-Sam/Ship2-Sam complex
6F7M MST S13ST Ship2-Sam/52.2±0.7 µM NA [94]
6F7N MST S13ST (short) Ship2-Sam/No binding NA [94]
6F7O MST A5ST Ship2-Sam/No binding NA [94]
Human Cul3-BTB 2MYL FP Cul349-68EN 620±177 NA [95]
2MYM FP Cul349-68LA 305±100 NA [95]
SIV 2JTP - RNA stem-loop - NA [96]
α-helical hairpin proteins
1EI0 - P8MTCP1 - NA [97]
De novo proteins 2M7C - Cp-T2C3b - NA [98]
2M7D - (P12W)-T2C16b - NA [98]
4.1 SAH-p53-8: stapled p53 peptide binds potently to human MDM2
p53, the main tumor suppressor, which is mainly negatively regulated by the E3 ubiquitin ligase MDM2. Although p53 is mutated or inactivated in more than 50% of human cancers, the other 50%
retain WT-p53. Therefore, the p53: MDM2 PPIs is a promising and confirmed target for drug
discovery and cancer therapy. This can be accomplished by discovering a potent MDM2 binder in
order to prevent its binding to p53 and thereby restore its biological function. In 2012, Baek and co-
workers were able to resolve a high-resolution structure of a stapled peptide inhibitor in complex
with MDM2 (SAH-p53-8 (PDB 3V3B)) [62]. This peptide was synthesized following ring-closing
olefin metathesis (RCM) at i,i+7 stapling positions between residues Asn20 and Leu26. As
anticipated by molecular dynamics (MD), the crystal structure revealed an extended region of the
helical peptide from residues 19-27 in the bound state that was not seen in other peptides with lower
affinities toward MDM2. Moreover, the bound peptide induced minor changes in MDM2,
specifically at the side chain of Met62 (which folds away from the p53 binding pocket, to make
space for the staple), Val93 (which shifts inside the binding pocket) and the side chain of Tyr100
that is found in a “closed” form. However, the α-helix peptide is located in the same position as the
native helix of p53, orienting the three residues critical for binding (Phe19, Trp23 and Leu26) in the
correct location (Figure 11). Remarkably, the aliphatic staple intimately interacts with the protein
and is located directly over the Met50−Lys64 helix and contributes ca. 10% of the peptide-Mdm2
total surface contact area. Additionally, the staple shields a H-bond between Trp23 and Leu54 from
solvent competition (Figure 12). Two novel features were discovered in the complex structure, first
an extended hydrophobic interface of the staple linker with Leu54, Phe55, Gly58, and Met62 of
Mdm2. The second feature is that the staple displaced a common water molecule present in most
MDM2 structures, which forms H-bonds with Gln59-N and Phe55-O. The later displacement likely
entropically stabilizes the complex during binding and contributes to SAH-p53-8 tight binding as
evidenced by an FP assay showing a K
dof 55nM. Lastly, stapling increases peptide helicity during
binding in relation to that of native p53 - influencing residue Leu26, which plays an important role
in MDM2 binding. Additionally, the researchers concluded that the long stapling i,i+7 enhanced
helical conformation and affinity as suggested by previous studies [19]. Subsequent to the discovery
of SAH-p53-8, several stapled peptides, such as sMTide-02 [99] and ASTP-7041 [35], showed
potent binding toward MDM2 with Kd values of 34.35 and 0.91nM, respectively. Additionally,
both peptides (in addition to VIP-84 (another stapled peptide targeting MDM2: p53)) showed
cellular permeability when tested using a nanoBRET (Bioluminescence Resonance Excitation
Transfer) live cell assay. Screening various lipid based formulations, the cellular uptake of VIP-84
was shown to be enhanced, as well as its biological activity, which was linked to vesicular or
endosomal escape of the stapled peptide through the cell membrane [100].
Figure 11: Alignment of the SAH-p53-8 peptide (yellow, PDB 3V3B) and the native p53 peptide (cyan, PDB 1YCR). The MDM2 molecule is shown in surface representation. SAH-p53-8 peptide mimics the three pharmacophore residues (Phe19, Leu26, Trp23) in the binding site in a similar manner to the native p53. The residues outside the Phe19- Leu26 regions are not visible, indicating conformational flexibility in the bound state. Moreover, the whole helix of stapled peptide moves by ~1Å and is rotated by 18°, allowing the Trp23 indole ring to form a hydrogen bond with MDM2 Leu54 (green line). Interestingly, Leu26 orientates itself in a distinct manner to that of the native p53 Leu26, (moving by 2.7 Å toward the N-terminus of the peptide) and the side chain is flipped by approximately 180° to fill the same pocket space. This feature is not found in any other reported structure.
.
Figure 12: A closer view of the SAH-p53-8 stapled peptide in a “closed” conformation state. The MDM2 molecule is shown in surface representation, the peptide (yellow) and the staple (orange) in sticks. A hydrogen bond is formed between the indole nitrogen atom of the peptide helix and the carbonyl oxygen of Leu54 of MDM2 (green line). This H-bound is protected from solvent competition by the staple that lied directly over Met50-Lys64 helix (the rim of p53 binding site). In addition, the staple intimately interacts with the protein surface and forms an extended hydrophobic interface with Leu54, Phe55, Gly58, and Met62 of Mdm2.
Leu26 Trp23
Phe19
Leu54
Leu54 Phe55
Gly58
Met62
4.2 MDM2 double macrocyclization stapled peptide: fast selection of cell-active inhibitor
Following on from the SAH-p53-8 potent peptide inhibitor, Lau et al. managed to synthesize a
stapled peptide-E1 by applying a novel stapling technique [64]. This technique is based on double
Cu-catalyzed azide–alkyne cycloaddition (CuAAC) and followed two-component strategies, in that
the staple and α-helix peptide are separated before cyclisation. This was combined with click
chemistry to generate a peptide with variable functional staples. The team used the p53-MDM2
interaction as a model, since that target has been well investigated as an oncogenic therapy for
cancers with overexpressed MDM2. For optimum inhibitor screening, and to ensure fast and easy
selection for the best peptide, cyclisation was conducted in situ and directly in primary cells
medium using a 96-well assay. This approach eliminated the extra purification step required in
other two-component strategies and provided a first example of stapling within a biological
environment. The first stapled peptide A1 was synthesized by linking diyne 1 to p53-derived
diazidopeptide A to produce A1 with 60% yield. Different peptide variants B-E were tested in situ
to define a peptide with the highest p53 activation, showing that the E+1 stapled peptide was the
most potent activator within cells. The binding affinity of the E1 peptide was measured using two
biophysical assays (FP and ITC) determining K
dvalues of 7.5±0.7 and 12±3 nM, respectively. The
crystal structure of E1-MDM2 (17-108, E69A/K70A) complex at 1.9Å resolution elucidated the
helical structure of E1 orienting the three hydrophobic key residues (Phe19, Trp23 and Leu26) in
the correct positioning for MDM2 binding (PDB 5AFG), in a manner broadly similar to that of the
p53 native peptide (Figure 13a). The bis (triazolyl) staple was discovered in an anti regioisomer and
four hydrophobic interactions were found with the protein surface residues: Leu54, Phe55, Gln59
and Met62. This mode of binding was similar to previously reported structure PDB: 3V3B
(described in section 4.1) indicating that both staples are sited at the rim area of the p53-binding
pocket, where Phe55 is the most important residue (Figure 13b). The proteolytic stability, cellular
uptake and toxicity of E1 peptide were evaluated, in which it showed high stability in a
chymotrypsin assay, significant cellular permeability observed by confocal microscopy and did not
show non-specific toxicity as determined in an LDH leakage assay.
Figure 13: The E1-MDM2 complex high-resolution structure at 1.9Å. a) top view of E1 stapled peptide (magenta, PDB 5AFG) aligned with the native peptide p53 (cyan, PDB 1YCR), revealing the typical mode of binding within the MDM2 hydrophobic pocket (grey surface) - placing the triad residues responsible for binding (P2he19, Trp23, Leu26) in the correct orientation to engage the MDM2 hotspots. The staple is found in anti regioisomer form and interacts with protein surface a similar mode as b) previously reported hydrocarbon SAH-p53-8 stapled peptide (PDB 3V3B), in that the stapled form four hydrophobic interactions with MDM2 surface residues (Leu54, Phe55, Gln59 and Met62, lime green), in which Phe55 is the most critical residue. The superimposition of the triazole-stapled E1 peptide with the correlated hydrocarbon-stapled p53 peptide (yellow, PDB 3V3B) suggests that both staples engage the same area that is located at the rim of the p53 binding pocket, on the Met50–Lys64 helix. The E1 stapled peptide is also shown in 2D for clarity (right).
4.3 Specific MCL-1 stapled peptide inhibitor as apoptosis sensitizer in cancer cells
The members of BCL-2 family known to have an anti-apoptotic role in cells are considered to be key pathogenic proteins in human diseases categorized by uncontrolled cell survival - such as cancer and autoimmune disorders. The MCL-1 protein belongs to this family and supports cell survival by trapping the apoptosis- inducing BCL-2 homology domain 3 (BH3) α-helix of pro- apoptotic BCL-2 family members. Cancer cells utilize this physiological phenomenon by overexpressing anti-apoptotic proteins to guarantee their immortality. As a result, developing an inhibitor to block the hydrophobic pocket of the anti-apoptotic proteins from binding the BH3 α- helix could lead to the discovery of a successful drug. By mimicking BH3 α-helix, several small molecules compounds were synthesized to inhibit anti-apoptotic proteins and some are undergoing clinical trials (including ABT-263, obatoclax, and AT-101). Most target three or more anti-
a)
Leu26 Phe19
Trp23
b) Gln59 Met62
Phe55
Leu54
N N N N N
N
NH C O NH C
O
EYWAQL S
LTF NH2
Ac
apoptotic member proteins, except the ABT-199 small molecule inhibitor, which has a high potency and specificity to the BCL-2 protein with a K
i< 0.010 nM. ABT-199 was discovered through reverse engineering of navitoclax and keeping similar hydrophobic interactions but modifying the electrostatic interaction with Arg103 (specific to BCL-2 not BCL-XL) [101]. Furthermore, ABT- 199 has antitumor activity against different cancers as non-Hodgkin’s lymphoma (NHL) [101], refractory chronic lymphocytic leukemia (CLL) [102,103], and BCL-2–dependent acute lymphoblastic leukemia (ALL) [101] in vitro. The same positive results were found in vivo when ABT-199 was tested on a wide spectrum of xenograft mouse models harboring human hematological tumor (RS4;11), B cell lymphoma with the t(14;18) translocation [101] and mantel cell lymphoma (MCL) [101,104].
Nonetheless, the topography of the binding groove and the amino acids residues involved in the protein interaction of BH3 helix determine the specificity of the anti-apoptotic protein-binding partner. Therefore, the need to discover an inhibitor that selectively targets the interacting surface, which is large and complex, is essential. Walensky and his group [68] selected MCL-1 as their research target, due to its survival role in a wide-range of cancers and protein overexpression that has been linked to the pathogenesis of diverse refractory cancers (including multiple myeloma, acute myeloid leukemia, melanoma and poor prognosis breast cancer [105–108]). This group was able to synthesize a highly potent stapled peptide (MCL-1 SAHB
D), which selectively binds MCL-1 and prevent it from suppressing the apoptosis pathway and sensitizing caspase-dependent apoptosis within cancer cells. A library of stapled alpha-helices of BCL-2 domain peptides was synthesized based on the BH3 domain of human BCL-2 family and stapling was located at the i,i+4 positions using ring-closing metathesis (RCM). To define the binding and specificity of BH3 helix and MCL- 1 alone, alanine scanning, site-direct mutagenesis and staple scanning were performed; the results indicated that MCL-1 SAHB
Dhas the highest helicity ~90% and strongest binding, with a K
dof 10 nM as determined by an FP assay. The complex structure of the stapled peptide with MCL-1ΔNΔC was solved at 2.3Å resolution (PDB 3MK8) and showed that MCL-1 SAHB
Dis present in a helical conformation and interacts with the MCL-1 canonical BH3-binding pocket. The peptide α-helix conserved residues L213, V216, G217, and V220 make a direct hydrophobic contact with the MCL- 1 interface that is consistent with many BH3 domains. These hydrophobic interactions are reinforced by a salt bridge between MCL-1 SAHB
DAsp218 and MCL-1ΔNΔC Arg263 (Figure 14).
Interestingly, the hydrocarbon staple with alkene cis conformation made a distinct hydrophobic contact with the edge of the MCL-1 binding site. Moreover, the methyl group explores a groove comprising Gly262, Phe318, and Phe319 of MCL-1 and additional contact was found between the staple aliphatic side chain and the edge of the main interaction site (Figure 15). All of these structural evidence indicate the role of the staple in the high affinity binding of the peptide and its ability to provide biological specificity toward MCL-1. This group also demonstrated the capacity of MCL-1 SAHB
Dto effectively sensitize mitochondrial apoptosis in vitro using wild type and Bak−/− mitochondria mouse models and in OPM2 cells by measuring the dissociation of native inhibitory MCL-1/BAK complexes using FP assay. In comparison to ABT-199, the MCL-1 SAHB
Dstapled peptide shows good cell permeability and has the capacity to sensitize cancer cells to
apoptosis when tested on Jurkat T-cell leukemia and OPM2 cells, underscoring the clinical
relevance of these findings. However, the MCL-1 stapled peptide has not yet been evaluated in clinical trials.
Figure 14: The crystal structure of MCL-1 SAHBD stapled peptide (slate helix) binding to the MCL-1ΔNΔC (light pink surface) interface at the canonical BH3-binding groove, solved at 2.3Å resolution (PDB 3MK8). The peptide makes several hydrophobic interactions, including the hydrophobic residues Leu213, Val216, Gly217, and Val220 of MCL-1 SAHBD making direct contact with a hydrophobic cleft at the surface of MCL- 1ΔNΔC (hot pink). The hydrophobic interaction are reinforced by a salt bridge between MCL-1 SAHBD Asp218 and MCL-1ΔNΔC Arg263 (blue) and these residues also contribute to a hydrogen bond cluster that includes MCL-1ΔNΔC Asp256 and Asn260 (green).
Figure 15: The hydrocarbon staple of MCL-1 SAHBD peptide with an alkene functionality in the cis conformation (yellow stick) makes distinct hydrophobic contacts with the MCL-1ΔNΔC binding site border (light pink surface). A methyl group of the α,α- dimethyl functionality engages with a groove consisting of MCL-1ΔNΔC Gly262, Phe318, and Phe319 residues (raspberry sticks).
Phe318 Phe319
Gly262
